The present invention relates to isolated DNA molecules encoding single strand gap response proteins involved in activation of a DNA repair/cell cycle checkpoint pathway, as well as diagnostic and therapeutic uses of the DNA molecules, their expressed proteins or polypeptides, and antibodies raised against the proteins or polypeptides.
The progression of a eukaryotic cell through the stages of the cell cycle can be arrested if the events of the previous stage of the cell cycle, such as DNA replication, have not been completed or, in addition, if the DNA has sustained some type of damage. The controls on cell cycle progression are termed checkpoints (Hartwell, L., et al., xe2x80x9cCheckpoints: Controls That Ensure the Order of Cell Cycle Events,xe2x80x9d Science, 246:629-34 (1989)), and they can be used to detect whether the processes of the individual stages of the cell cycle have been completed and whether the DNA is intact or in need of repair. Genes whose expressed products are involved in cell cycle delay or DNA repair are broadly defined as checkpoint control genes. Cells that are mutated in one of the cell cycle checkpoint control genes, however, are able to proceed from one stage of the cell cycle to the next even if the cellular processes of that stage are incomplete or in the presence of DNA damage. The G2 phase of the cell cycle lies between S phase, in which DNA replication takes place, and M phase, when mitosis occurs. Thus, the G2 checkpoint is critical for ensuring that mitosis does not occur until all the necessary steps of DNA replication, DNA repair, and chromosome duplication are complete.
Many checkpoint-deficient mutants have been identified in the budding yeast Saccharomyces cerevisiae and in the fission yeast Schizosaccharomyces pombe. 
Genes have been isolated that link mitosis to the completion of DNA replication. Enoch, T., et al., xe2x80x9cMutation of Fission Yeast Cell Cycle Control Genes Abolishes Dependence of Mitosis on DNA Replication,xe2x80x9d Cell, 60:665-73 (1990); Enoch, T., et al., xe2x80x9cFission Yeast Genes Involved in Coupling Mitosis to Completion of DNA Replication,xe2x80x9d Genes Dev., 6:2035-46 (1992); McFarlane, R. J., et al., xe2x80x9cCharacterization of the Schizosaccharomyes pombe rad4/cut5 Mutant Phenotypes: Dissection of DNA Replication and G2 Checkpoint Control Function,xe2x80x9d Mol. Gen. Genet., 255:332-40 (1997). In addition, many genes that function in DNA repair have been identified as G2 checkpoint control genes. Nasim, A., et al., xe2x80x9cGenetic Control of Radiation Sensitivity in Schizosaccharomyces pombe,xe2x80x9d Genetics, 79:573-82 (1975); Al-Khodairy, F., et al., xe2x80x9cDNA Repair Mutants Defining G2 Checkpoint Pathways in Schizosaccharomyces pombe,xe2x80x9d EMBO J., 11:1343-50 (1992); Al-Khodairy, F., et al., xe2x80x9cIdentification and Characterization of New Elements Involved in Checkpoint and Feedback Controls in Fission Yeast,xe2x80x9d Mol. Biol. Cell, 5:147-60 (1994). Several examples include Saccharomyces cerevisiae RAD9 (Weinert, T. A., et al., xe2x80x9cCharacterization of RAD9 of Saccharomyces cerevisiae and Evidence that Its Function Acts Post-Translationally in Cell Cycle Arrest after DNA Damage,xe2x80x9d Mol. Cell. Biol., 10:6554-64 (1990)), Saccharomyces cerevisiae MEC3 (Weinert, T. A., et al., xe2x80x9cMitotic Checkpoint Genes in Budding Yeast and the Dependence of Mitosis on DNA Replication and Repair,xe2x80x9d Genes and Dev., 8:652-65 (1994)), Schizosaccharomyces pombe rad1 (Rowley, R., et al., xe2x80x9cCheckpoint Controls in Schizosaccharomyces pombe: rad1, xe2x80x9d EMBO J., 11:1335-42 (1992)), Schizosaccharomyces pombe rad3 (Jimenez, G., et al., xe2x80x9cThe rad3+ Gene of Schizosaccharomyces pombe is Involved in Multiple Checkpoint Functions and in DNA Repair,xe2x80x9d Proc. Natl. Acad. Sci. USA, 89:4952-56 (1992); Bentley, N. J., et al., xe2x80x9cThe Schizosaccharomyces pombe rad3 Checkpoint Gene,xe2x80x9d EMBO J., 15:6641-51 (1996)), Schizosaccharomyces pombe rad17 (Griffiths, D. J. F., et al., xe2x80x9cFission Yeast rad17: a Homolog of Budding Yeast RAD24 That Shares Regions of Sequence Similarity with DNA Polymerase Accessory Proteins,xe2x80x9d EMBO J., 14:5812-23 (1995)), Schizosaccharomyces pombe hus1 (Kostrub, C. F., et al., xe2x80x9cMolecular Analysis of hus1+, a Fission Yeast Gene Required for S-M and DNA Damage Checkpoints,xe2x80x9d Mol. Gen. Genet., 254:389-99 (1997)), and the fungus Ustilago maydis REC1 (One1, K., et al., xe2x80x9cThe REC1 Gene of Ustilago maydis. Which Encodes a 3xe2x80x2-5xe2x80x2 Exonuclease, Couples DNA Repair and Completion of DNA Synthesis to a Mitotic Checkpoint,xe2x80x9d Genetics, 143:165-74 (1996)). A number of reviews summarize this work. Sheldrick, K. S., et al., xe2x80x9cFeedback Controls and G2 Checkpoints: Fission Yeast as a Model System,xe2x80x9d BioEssays, 15:775-82 (1993); Lydall, D., et al., xe2x80x9cFrom DNA Damage to Cell Cycle Arrest and Suicide: A Budding Yeast Perspective,xe2x80x9d Curr. Opin. Genet. Dir. 6:4-11 (1996); Stewart, E., et al., xe2x80x9cS-phase and DNA-damage Checkpoints: a Tale of Two Yeasts,xe2x80x9d Curr. Opin. Cell Biol., 8:781-87 (1996); Carr, A. M., xe2x80x9cControl of Cell Cycle Arrest by the Mec1sc/Rad3sp DNA Structure Checkpoint Pathway,xe2x80x9d Curr. Opin. Genet. Dev., 7:93-98 (1997). Some of the Saccharomyces cerevisiae and Schizosaccharomyces pombe genes involved in the G2 cell cycle checkpoint are summarized in FIG. 1.
A human homolog of the Schizosaccharomyces pombe rad9 checkpoint control gene was described recently. Lieberman, H. B., et al., xe2x80x9cA Human Homolog of the Schizosaccharomyces pombe rad9+ Checkpoint Control Gene,xe2x80x9d Proc. Natl. Acad. Sci. USA, 93:13890-95 (1996). The mapping of a human homolog to Schizosaccharomyces pombe rad1 was reported. Parker, A., et al., xe2x80x9cIdentification of a Putative Human Homolog of the Schizosaccharomyces pombe rad1 Checkpoint Genexe2x80x9d, Eukayrotic DNA Replication, p. 179, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1997). Interestingly, referring to FIG. 1, two Saccharomyces cerevisiae genes, MEC3 and RAD9, do not appear to have Schizosaccharomyces pombe or human homologs, while Saccharomyces cerevisiae does not carry homologs for Schizosaccharomyces pombe hus1 or rad9. The checkpoint control systems of Saccharomyces cerevisiae and Schizosaccharomyces pombe have some gene homologs in common; however, they appear to have diverged significantly, perhaps because cell division is so different in these organisms. Saccharomyces cerevisiae divides by budding, while Schizosaccharomyces pombe divides by fission. The mitosis and cell division of Schizosaccharomyces pombe is much more similar to that of human cells than the mitosis and cell division of Saccharomyces cerevisiae. Schizosaccharomyces pombe has a distinct G2 phase of the cell cycle and, in addition, the chromosomes undergo condensation during mitosis. Russell, P., et al., xe2x80x9cSchizosaccharomyces pombe and Saccharomyces cerevisiae: A Look at Yeasts Divided,xe2x80x9d Cell, 45:781-82 (1986). This may be why the human genes of the G2 cell cycle checkpoint pathway correspond so much more closely to the genes of Schizosaccharomyces pombe than to the genes of Saccharomyces cerevisiae. 
The occurrence of mutations in checkpoint control genes of higher eukaryotes can lead to cancer. Hartwell, L., xe2x80x9cDefects in a Cell Cycle Checkpoint may be Responsible for the Genomic Instability of Cancer Cells,xe2x80x9d Cell, 71:543-46 (1992); Hartwell, L., et al., xe2x80x9cCell Cycle Control and Cancer,xe2x80x9d Science, 266:1821-28 (1994); Kastan, M. B., et al., xe2x80x9cParticipation of p53 Protein in the Cellular Response to DNA Damage,xe2x80x9d Cancer Res., 51:6304-11 (1991); Kuerbitz, S. J., et al., xe2x80x9cWild-Type p53 is a Cell Cycle Checkpoint Determinant Following Irradiation,xe2x80x9d Proc. Natl. Acad. Sci. USA, 89:7491-95 (1992). Genes which, when mutated, allow increased rates of tumor formation are termed tumor suppressors. Many tumor suppressors have cell cycle checkpoint function, and loss-of-function mutations in these genes causes runaway cell proliferation, leading to tumor formation. Collins, K., et al., xe2x80x9cThe Cell Cycle and Cancer,xe2x80x9d Proc. Natl. Acad. Sci. USA, 94:2776-78 (1997). For example, ATM has been identified as the gene that is defective in patients with ataxia telangiectasia. As seen in FIG. 1, ATM is the human homolog of Saccharomyces cerevisiae MEC1/ESR1 and Schizosaccharomyces pombe rad3. Nowak, R., xe2x80x9cDiscovery of AT Gene Sparks Biomedical Research Bonanza,xe2x80x9d Science, 268:1700-01 (1995); reviewed by Enoch, T., et al., xe2x80x9cCellular Responses to DNA Damage: Cell-Cycle Checkpoints, Apoptosis and the Roles of p53 and ATM,xe2x80x9d Trends Biochem. Sci., 20:426-30 (1995); Lehmann, A. R., et al., xe2x80x9cThe Ataxia-Telangiectasia Gene: a Link Between Checkpoint Controls, Neurodegeneration, and Cancer,xe2x80x9d Trends Genet., 11:375-77 (1995); Jackson, S. P., xe2x80x9cThe Recognition of DNA Damage,xe2x80x9d Curr. Opin. Genet. Dev., 6:19-25 (1996). These proteins have protein kinase activity and are involved in generating a signal to halt progression through the cell cycle in response to DNA damage.
The genes of G2 cell cycle checkpoint function in a number of cellular contexts in both Schizosaccharomyces pombe and Saccharomyces cerevisiae. One such function involves causing a G2 phase-specific cell cycle arrest in response to DNA damage from UV or gamma-irradiation, thereby blocking the onset of mitosis. In this instance, they are responding to DNA damage-specific structures. In Schizosaccharomyces pombe , this is mediated by the Chk1 protein kinase. Walworth, N. C., et al. xe2x80x9crad-Dependent Response of the ChK1-Encoded Protein Kinase at the DNA Damage Checkpoint,xe2x80x9d Science, 271:353-56 (1996). Another function involves causing a delay of S-phase and allowing S-phase recovery in response to stalled DNA replication, as can be induced by exposure of cells to hydroxyurea. In this case, the genes are responding to a DNA replication-specific structure. In Schizosaccharomyces pombe, this is mediated by the Cds1 protein kinase. Lindsay, H. M., et al., xe2x80x9cS-Phase-Specific Activation of Cds1 Kinase Defines a Subpathway of the Checkpoint Response in Schizosaccharomyces pombe,xe2x80x9d Genes Div. 12:382-95 (1998). Finally, in Saccharomyces cerevisiae, these genes induce a G2 arrest upon inactivation of a cdc13 temperature-sensitive mutant. Lydall, D., et. al., xe2x80x9cYeast Checkpoint Genes in DNA Damage Processing: Implications for Repair and Arrest,xe2x80x9d Science, 270:1488-91 (1995). Inactivation of cdc13 results in the appearance of single stranded TG-rich regions at telomeres due to a specific loss of the AC-rich DNA strands.
The unifying principle in these instances is activation of the cell cycle checkpoint pathway by regions of single stranded DNA. After DNA damage, such as by UV or gamma-irradiation, a single strand gap is created by excision of a length of DNA containing the damaged nucleotides. Sancar, A., xe2x80x9cExcision Repair in Mammalian Cells,xe2x80x9d J. Biol. Chem., 270:15915-18 (1995). The stalling of DNA replication forks will normally result in a single strand region on the lagging strand, even if the leading strand is fully replicated up to the point at which the parental DNA strands have not been unwound and remain duplexed. Impaired functioning of Saccharomyces cerevisiae cdc13 renders the telomeric TG strand single stranded, perhaps due to a reduced protection of the CA strand from degradation. Garvik, B., et al., xe2x80x9cSingle-Stranded DNA Arising at Telomeres in cdc13 Mutants May Constitute a Specific Signal for the RAD9 Checkpoint,xe2x80x9d Mol. Cell. Biol., 15:6128-38 (1995); Nugent, C. I., et al., xe2x80x9cCdc13p: A Single-Strand Telomeric DNA-Binding Protein with a Dual Role in Yeast Telomere Maintenance,xe2x80x9d Science, 274:249-52 (1996); Lin, J. J., et al., xe2x80x9cThe Saccharomyces CDC13 Protein is a Single-Strand TGI1-3 Telomeric DNA-binding Protein in vitro that Affects Telomere Behavior in vivo,xe2x80x9d Proc. Natl. Acad. Sci. USA, 93:13760-65 (1996). Thus, these specific checkpoint control genes appear to monitor the intactness of cellular DNA by responding to the generation of single stranded regions. They act in concert with one another to slow S phase or induce G2 arrest when single stranded gaps appear in the DNA. As such, they constitute genes of a distinct pathway of the G2 cell cycle checkpointxe2x80x94i.e. the xe2x80x9csingle strand gap responsexe2x80x9d (SSGR) pathway. Genes involved in the SSGR pathway are specifically defined as xe2x80x9csingle strand gap responsexe2x80x9d (SSGR) genes.
Researchers have isolated and sequenced several SSGR genes in Schizosaccharomyces pombe and Saccharomyces cerevisiae. 
The gene for Schizosaccharomyces pombe rad17 has been described in Griffiths, D. J. F., et al., xe2x80x9cFission Yeast rad17: a Homolog of Budding Yeast RAD24 That Shares Regions of Sequence Similarity with DNA Polymerase Accessory Proteins,xe2x80x9d EMBO J., 14:5812-23 (1995). The gene for its homolog in Saccharomyces cerevisiae, RAD24, has been deposited in EMBL/Genbank/DDBJ data banks, which are publicly available databases. Zhu, Y. B., et al., xe2x80x9cMolecular Cloning and Sequencing of DNA Repair Gene RAD24, Chinese Biochem. J., 11:541-50 (1995). Cloning of Schizosaccharomyces pombe rad17 revealed that it has extensive homology to the DNA polymerase accessory proteins known as clamp loaders. Griffiths, D. J. F., et al., xe2x80x9cFission Yeast rad17: a Homolog of Budding Yeast RAD24 That Shares Regions of Sequence Similarity with DNA Polymerase Accessory Proteins,xe2x80x9d EMBO J., 14:5812-23 (1995). This suggests that Schizosaccharomyces pombe rad17 may carry out a clamp loading or unloading function in the DNA repair pathway. Interestingly, while the Schizosaccharomyces pombe rad17 gene carries out two roles, DNA repair and cell cycle checkpoint regulation, the two functions, are separable. Specific point mutations of rad17 were generated that abolished DNA repair activity but did not affect checkpoint control. Griffiths, D. J. F., et al., xe2x80x9cFission Yeast rad17: a Homolog of Budding Yeast RAD24 That Shares Regions of Sequence Similarity with DNA Polymerase Accessory Proteins,xe2x80x9d EMBO J., 14:5812-23 (1995).
The cloning of the gene for Schizosaccharomyces pombe rad1 has been described in a series of reports. Sunnerhagen, P., et al., xe2x80x9cCloning and Analysis of a Gene Involved in DNA Repair and Recombination, the rad1 Gene of Schizosaccharomyces pombe,xe2x80x9d Mol. Cell. Biol., 10:3750-60 (1990); Rowley, R., et al., xe2x80x9cCheckpoint Controls in Schizosaccharomyces pombe: rad1,xe2x80x9d EMBO J., 11:1335-42 (1992); Long, K. E., et al., xe2x80x9cThe Schizosaccharomyces pombe rad1 Gene Consists of Three Exons and the cDNA Sequence is Partially Homologous to the Ustilago maydis REC1 cDNA,xe2x80x9d Gene, 148:155-59 (1994). The cloning of its Saccharomyces cerevisiae homolog, RAD17, has also been described in Siede, W., et al., xe2x80x9cCloning and Characterization of RAD17, a Gene Controlling Cell Cycle Responses to DNA Damage in Saccharomyces cerevisiae,xe2x80x9d Nuc. Acids Res., 24:1669-75 (1996). Extensive work has been carried out over a number of years concerning its homolog in Ustilago maydis, REC1. Holliday, R., et al., xe2x80x9cGenetic Characterization of rec-1, a Mutant of Ustilago maydis Defective in Repair and Recombination,xe2x80x9d Genet. Res., 27:413-53 (1976); Holden, D. W., et al., xe2x80x9cNucleotide Sequence of the REC1 Gene of Ustilago maydis,xe2x80x9d Nuc. Acids Res., 17:10489 (1989); Tsukuda, T., et al., xe2x80x9cIsolation of the REC1 Gene Controlling Recombination in Ustilago maydis,xe2x80x9d Gene, 85:335-41 (1989). An exonuclease activity is associated with the protein (Thelen, M. P., et al., xe2x80x9cThe REC1 Gene of Ustilago maydis Involved in the Cellular Response to DNA Damage Encodes an Exonuclease,xe2x80x9d J. Biol. Chem., 269:747-54 (1994)), and the role of the gene in DNA repair and cell cycle regulation is known (Onel, K., et al., xe2x80x9cMutation Avoidance and DNA Repair Proficiency in Ustilago maydis Are Differentially Lost with Progressive Truncation of the REC1 Gene Product,xe2x80x9d Mol. Cell. Biol., 15:5329-38 (1995); Onel, K., et al., xe2x80x9cThe REC1 Gene of Ustilago maydis, Which Encodes a 3xe2x80x2-5xe2x80x2 Exonuclease, Couples DNA Repair and Completion of DNA Synthesis to a Mitotic Checkpoint,xe2x80x9d Genetics, 143:165-74 (1996)).
Recent studies offer some insight into the in vivo role of the Saccharomyces cerevisiae checkpoint genes. Lydall D., et al., xe2x80x9cYeast Checkpoint Genes in DNA Damage Processing: Implications for Repair and Arrest,xe2x80x9d Science, 270:1488-91 (1995). The Rad24, Rad17, and Mec3 proteins appear to activate an exonuclease activity in vivo, while the Rad9 protein appears to modulate exonuclease activity. It was suggested that Saccharomyces cerevisiae Rad 17 protein may actually be an exonuclease, based on homology between it and U. maydis Rec1.
The cloning of Schizosaccharomyces pombe hus1 was described recently. Kostrub, C. F., et al., xe2x80x9cMolecular Analysis of hus1+, a Fission Yeast Gene Required for S-M and DNA Damage Checkpoints,xe2x80x9d Mol. Gen. Genet., 254:389-99 (1997). Yeast strains disrupted in hus1 are viable but are checkpoint-defective.
It is expected that the mammalian homologs of the SSGR genes of Schizosaccharomyces pombe will also carry out an SSGR function in human cells, and are likely candidates for human tumor suppressor genes. However, no mammalian, particularly human, SSGR genes have been cloned. The present invention is directed to overcoming this deficiency in the art.
The present invention relates to isolated DNA molecules encoding mammalian xe2x80x9csingle strand gap responsexe2x80x9d (SSGR) proteins involved in activation of a DNA repair/cell cycle checkpoint pathway. In particular, the isolated DNA molecules include the human HRAD17, human HRAD1, human HHUS1, mouse HRAD1, and mouse HHUS1 DNA molecules. The present invention is also directed to proteins or polypeptides encoded by the DNA molecules as well as antibodies raised against those proteins or polypeptides. Expression systems and host cells transformed with these DNA molecules are also disclosed.
The DNA molecules of the present invention are useful in detecting the presence of genes encoding SSGR proteins in a tissue sample. This may be useful in identifying the presence of mutants in germline tissue samples, which indicates the potential for development of inheritable diseases, and in tumor samples, which indicates the particular mutation attributable to tumor formation and, therefore, may identify suitable treatment regimen.
The DNA molecules of the present invention can also be used in gene therapy to restore proper cell cycle regulation to cells. This is particularly useful in halting or reversing a cancerous or pre-cancerous condition.
The proteins or polypeptides of the present invention can be utilized to detect the presence of antibodies raised by such proteins or polypeptides in a sample of mammalian origin.
The antibodies or binding portions thereof of the present invention are useful for detecting the presence or absence of the expressed SSGR proteins or polypeptides from a sample.
The present invention also relates to a transgenic animal whose somatic and germ cells lack a gene encoding a SSGR protein or possess a disruption in that gene. This animal is susceptible to spontaneous tumor development and, therefore, is useful for studying tumor formation and treatment.
As tumor supressors, the SSGR proteins act at the G2 cell cycle checkpoint in order to permit complete repair of DNA damage. Their mutation leads to cancer by allowing increased amounts of DNA damage, leading to increased levels of mutation in the cell. Therefore, one benefit of the invention is its use as a tool for the diagnosis of cancers caused by mutations of these genes.
However, there is a second reason for the importance of the SSGR genes with respect to cancer, aside from their role as tumor supressors, and that is as a crucial consideration for chemotherapy. These genes function at the G2 cell cycle checkpoint and act as a barrier to the initiation of mitosis in the presence of unrepaired DNA damage. This barrier allows a cell with DNA damage to halt the cell cycle at the G2 stage for up to 8 hours, or more, and make repeated attempts to repair the DNA damage before allowing mitosis to proceed. This is useful to the cell because entry into mitosis in the presence of high levels of unrepaired DNA damage causes mitotic catastrophe and cell death (e.g., apoptosis). As such, this pathway is of key importance in the problematic survival of cancerous tissue or tumor cells after they are assaulted by chemotherapeutic DNA-damaging agents or radiotherapy.
Since cancers with mutations in the SSGR genes will have an increased susceptibility to DNA-damaging chemotherapeutic agents, it is important to determine whether the SSGR genes are mutated. Identification of cancers carrying mutations in or causing reduced expression of these genes would flag those cancers as being particularly susceptible to DNA-damaging chemotherapeutic agents, as opposed to drugs that poison the cell in other ways.
Furthermore, the SSGR proteins are key targets for anti-cancer drugs in those cancers that do not carry mutations in the SSGR genes. Inhibition of SSGR response protein function in these cancers will heighten the potency of chemotherapeutic DNA-damaging agents. The proteins or polypeptides of the present invention can be used to identify drugs that will inhibit the SSGR pathway and increase the effectiveness of tumor destruction by DNA-damaging agents.